Selective fishing and analysis of xanthine oxidase binders from two Fabaceae species by coupling enzyme functionalized core–shell magnetic nanoparticles with HPLC–MS

Xanthine oxidase (XOD) immobilized core–shell magnetic silica (Fe3O4@SiO2-XOD) nanoparticles coupled with high performance liquid chromatography–mass spectrometry (HPLC–MS) was developed to fish out and analyze XOD binders from two Fabaceae species, Puerariae lobata flower and Glycyrrhiza uralensis root. The prepared Fe3O4@SiO2-XOD nanoparticles exhibited good specificity for XOD binders, better dispersion in aqueous solution and reusability than those of Fe3O4-XOD nanoparticles. The amount of XOD immobilized onto Fe3O4@SiO2 nanoparticles was 339.9 μg/mg and the activity of Fe3O4@SiO2-XOD nanoparticles remained 95% after ten times usage. The optimum conditions of selective fishing were optimized, and finally incubating pH was set at 7, incubating temperature at 25 °C and adsorption time at 30 min. Twelve XOD binders were successfully identified from ethyl acetate extract of P. lobata flower and G. uralensis root. The developed method provides a rapid, purposeful and effective way to identify active compounds from natural complex mixtures.